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Publication Number
US-10407677-B2
Publication Date
2019-09-10
Expiration Date
Abstract
The present invention relates to a method for producing pools of siRNA molecules suitable for RNA interference.
Core Innovation
The invention relates to preparing different double stranded RNA molecules for target specific RNA interference, where each strand has a length of 15 to 30 nucleotides. The method uses in vitro transcription from custom DNA templates to generate RNA molecules that include a target-sequence-element and a loop-sequence-element, and the RNA molecules are hybridized to obtain a double stranded RNA molecule comprising single stranded loop-sequence-elements.
A defining feature is that the loop-sequence-elements are not complementary to the target gene and are selected to be cleavable by RNase T1. The hybridized RNA includes single stranded RNA loops that RNase T1 preferentially recognizes, cleaves, and digests to remove the single stranded loops, providing short double stranded RNA molecules in which each strand remains 15 to 30 nucleotides and can mediate target-specific RNA interference of at least one target gene.
The DNA template design pairs two DNA molecules that encode reverse-complement target-sequence-elements and non-reverse-complement loop-sequence-elements. The first DNA molecule contains repeating units of (target-sequence-element)-(loop-sequence-element), while the second DNA molecule contains repeating units of (target-sequence-element rc)-(loop-sequence-element), and the loop-sequence-elements are selected from 5′-AGTTG-3′ and 5′-AGTTTG-3′ such that no hybridization occurs between molecules consisting just of the loop-sequence-element.
The disclosed approach is applied to produce high-complexity siRNA pools (siPools) using in vitro transcription from custom DNA templates that generate annealed RNA precursors with double-stranded siRNA regions separated by RNase-cleavable single-stranded loop regions. The short dsRNA products are reported to yield functional RNA interference, and complex pools are described as improving on-target knockdown while substantially reducing off-target effects compared with known off-target individual siRNAs, smart pools, and esiRNAs.
Claims Coverage
The provided material identifies one independent claim that defines a method for preparing different double stranded RNA molecules producing short dsRNA (15–30 nucleotides per strand) capable of target specific RNA interference. The claim includes six inventive features that collectively define DNA template design, in vitro transcription, hybridization into dsRNA with single stranded loops, and RNase T1 digestion to remove loops and yield the final short dsRNA.
Target-sequence-element and loop-sequence-element repeating DNA templates
Providing at least one first DNA molecule with repeating units of (target-sequence-element)-(loop-sequence-element) where the target-sequence-element is a continuous sequence of 15 to 30 deoxyribonucleotides identical to a sequence in the target gene and the loop-sequence-element is a continuous sequence of 5 to 14 deoxyribonucleotides not complementary to the target gene.
Reverse-complement target pairing with non-complementary loops
Providing at least one second DNA molecule with repeating units of (target-sequence-element rc)-(loop-sequence-element) where the target-sequence-elements rc reverse-complement the target-sequence-elements from the first DNA molecule and the loop-sequence-elements in the second DNA molecule are not reverse complements of the loop-sequence-elements in the first DNA molecule.
In vitro transcription from first and second DNA molecules
In vitro transcribing the first and second DNA molecules using an RNA polymerase to obtain first and second RNA molecules.
Hybridizing RNAs to form dsRNA with single stranded loop-sequence-elements
Hybridizing the first and second RNA molecules to obtain a double stranded RNA molecule comprising single stranded loop-sequence-elements.
RNase T1 cleavage of single stranded loop regions to generate short dsRNA
Digesting the double stranded RNA molecule with RNase T1 that preferentially recognizes, cleaves, and digests the single stranded loop-sequence-elements to remove RNA loops and provide short double stranded RNA molecules where each strand has a length of 15 to 30 nucleotides capable of target-specific RNA interference.
Loop sequence selection for RNase T1 cleavage and no loop-only hybridization
Selecting each loop-sequence-element to be cleavable by RNase T1 and to have a sequence selected from 5′-AGTTG-3′ and 5′-AGTTTG-3′ such that there is no hybridization of two molecules consisting just of the loop-sequence-element.
Across the independent claim, the inventive core is the paired DNA template architecture that encodes reverse-complement target-sequence-elements and non-reverse-complement loop-sequence-elements, followed by in vitro transcription, hybridization to form dsRNA with single stranded loops, and RNase T1 digestion of cleavable loops to yield short 15–30 nucleotide per strand dsRNA that mediates target-specific RNA interference.
Stated Advantages
Provides short double stranded RNA molecules capable of target-specific RNA interference of at least one target gene.
RNase T1 preferentially cleaves the single stranded loop-sequence-elements to remove single stranded RNA loops, yielding short double stranded RNA molecules with each strand of 15 to 30 nucleotides.
Loop-sequence-elements are selected so that there is no hybridization of two molecules consisting just of the loop-sequence-element.
Improves on-target knockdown.
Substantially reduces off-target effects compared with known off-target individual siRNAs, smart pools, and esiRNAs.
Documented Applications
Targeting AURKB (AUKRB), Scyl1, and PolG, with Mad2 off-target monitoring and functional demonstration readouts including qPCR/RT-PCR, luciferase reporters, Western blot, and global expression analysis.
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